Throughout this application, various publications are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these publications may be found at the end of the specification, preceding the sequence listing and the claims.
Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY and its relatives (peptide YY or PYY, and pancreatic polypeptide or PP) elicit a broad range of physiological effects through activation of at least five G protein-coupled receptor subtypes known as Y1, Y2, Y3, Y4 (or PP), and the xe2x80x9catypical Y1xe2x80x9d. The role of NPY as the most powerful stimulant of feeding behavior yet described is thought to occur primarily through activation of the hypothalamic xe2x80x9catypical Y1xe2x80x9d receptor. This receptor is unique in that its classification was based solely on feeding behavior data, rather than radioligand binding data, unlike the Y1, Y2, Y3, and Y4 (or PP) receptors, each of which were described previously in both radioligand binding and functional assays.
The peptide neurotransmitter neuropeptide Y (NPY) is a 36 amino acid member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY is considered to be the most powerful stimulant of feeding behavior yet described (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Direct injection into the hypothalamus of satiated rats, for example, can increase food intake up to 10-fold over a 4-hour period (Stanley et al., 1992). The role of NPY in normal and abnormal eating behavior, and the ability to interfere with NPY-dependent pathways as a means to appetite and weight control, are areas of great interest in pharmacological and pharmaceutical research (Sahu and Kalra, 1993; Dryden et al., 1994). Any credible means of studying or controlling NPY-dependent feeding behavior, however, must necessarily be highly specific as NPY can act through at least 5 pharmacologically defined receptor subtypes to elicit a wide variety of physiological functions (Dumont et al., 1992). It is therefore vital that knowledge of the molecular biology and structural diversity of the individual receptor subtypes be understood as part of a rational drug design approach to develop subtype selective compounds. A brief review of NPY receptor pharmacology is summarized below and also in Table 1.
Rank orders of affinity for key peptides (NPY, PYY, PP, [Leu31, Pro34]NPY, NPY2-36, and NPY13-36) are based on previously reported binding and functional data (Schwartz et al., 1990; Wahlestedt et al., 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). Data for the Y2 receptor were disclosed in PCT International Application No. PCT/US95/01469, filed Feb. 3, 1995, International Publication No. WO 95/21245, published Aug. 10, 1995 the foregoing contents of which are hereby incorporated by reference. Data for the Y4 receptor were disclosed in PCT International Application No. PCT/US94/14436 filed Dec. 28, 1994, International Publication No. WO 95/17906, published Aug. 10, 1995 the contents of which are hereby incorporated by reference. Missing peptides in the series reflect a lack of published information. Table 1 reflects current information obtained with cloned human Y1, Y2, Y4, and Y5 receptors.
NPY receptor pharmacology has historically been based on structure/activity relationships within the pancreatic polypeptide family. The entire family includes the namesake pancreatic polypeptide (PP), synthesized primarily by endocrine cells in the pancreas; peptide YY (PYY), synthesized primarily by endocrine cells in the gut; and NPY, synthesized primarily in neurons (Michel, 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). All pancreatic polypeptide family members share a compact structure involving a xe2x80x9cPP-foldxe2x80x9d and a conserved C-terminal hexapeptide ending in Tyr36 (or Y36 in the single letter code). The striking conservation of Y36 has prompted the reference to the pancreatic polypeptides"" receptors as xe2x80x9cY-typexe2x80x9d receptors (Wahlestedt et al., 1987), all of which are proposed to function as seven transmembrane-spanning G protein-coupled receptors (Dumont et al., 1992).
The Y1 receptor recognizes NPYxe2x89xa7PYY greater than  greater than PP (Grundemar et al., 1992). The receptor requires both the N- and the C-terminal regions of the peptides for optimal recognition. Exchange of Gln34 in NPY or PYY with the analogous residue from PP (Pro34), however, is well-tolerated. The Y1 receptor has been cloned from a variety of species including human, rat and mouse (Larhammar et al, 1992; Herzog et al, 1992; Eva et al, 1990; Eva et al, 1992). The Y2 receptor recognizes PYYxcx9cNPY greater than  greater than PP and is relatively tolerant of N-terminal deletion (Grundemar et al., 1992). The receptor has a strict requirement for structure in the C-terminus (Arg33-Gln34-Arg35-Tyr36-NH2); exchange of Gln34 with Pro34, as in PP, is not well tolerated. The Y2 receptor has recently been cloned. The Y3 receptor is characterized by a strong preference for NPY over PYY and PP (Wahlestedt et al., 1991). [Pro34]NPY is reasonably well tolerated even though PP, which also contains Pro34, does not bind well to the Y3 receptor. The Y3 receptor (Y3) has not yet been cloned. The Y4 receptor binds PP greater than PYY greater than NPY. Like the Y1, the Y4 requires both the N- and the C-terminal regions of the peptides for optimal recognition. The xe2x80x9catypical Y1xe2x80x9d or xe2x80x9cfeedingxe2x80x9d receptor was defined exclusively by injection of several pancreatic polypeptide analogs into the paraventricular nucleus of the rat hypothalamus which stimulated feeding behavior with the following rank order: NPY2-36xe2x89xa7NPYxcx9cPYYxcx9c[Leu31, Pro34)NPY greater than NPY13-36 (Kalra et al., 1991; Stanley et al., 1992). The profile is similar to that of a Y1-like receptor except for the anomalous ability of NPY2-36 to stimulate food intake with potency equivalent or better than that of NPY. A subsequent report in J. Med. Chem. by Balasubramaniam et al. (1994) showed that feeding can be regulated by [D-Trp32]NPY. While this peptide was presented as an NPY antagonist, the published data at least in part support a stimulatory effect of [D-Trp32]NPY on feeding. [D-Trp32]NPY thereby represents another diagnostic tool for receptor identification. In contrast to other NPY receptor subtypes, the xe2x80x9cfeedingxe2x80x9d receptor has never been characterized for peptide binding affinity in radioligand binding assays and the fact that a single receptor could be responsible for the feeding response has been impossible to validate in the absence of an isolated receptor protein; the possibility exists, for example, that the feeding response could be a composite profile of Y1 and Y2 subtypes.
This invention now reports the isolation by expression cloning of a novel Y-type receptor from a rat hypothalamic cDNA library, along with its pharmacological characterization, in situ localization, and human homolog. The data provided link this newly-cloned receptor subtype, from now on referred to as the Y5 subtype, to the xe2x80x9catypical Y1xe2x80x9d feeding response. This discovery therefore provides a novel approach, through the use of heterologous expression systems, to develop a subtype selective antagonist for obesity and other indications.
This invention is based on the use of a 125I-PYY-based expression cloning technique to isolate a rat hypothalamic cDNA encoding an xe2x80x9catypical Y1xe2x80x9d receptor referred to herein as the Y5 receptor subtype. This application also concerns the isolation and characterization of a Y5 homolog from human hippocampus.
Protein sequence analysis reveals that the Y5 receptor belongs to the G protein- coupled receptor superfamily. Both the human and rat homolog displayxe2x89xa642% identity in transmembrane domains with the previously cloned xe2x80x9cY-typexe2x80x9d receptors. Rat brain localization studies using in situ hybridization techniques verified the existence of Y5 receptor mRNA in rat hypothalamus. Pharmacological evaluation revealed the following similarities between the Y5 and the xe2x80x9catypical Y1xe2x80x9d receptor. 1) Peptides bound to the Y5 receptor with a rank order of potency identical to that described for the feeding response: NPYxe2x89xa7NPY2-36=PYY=[Leu31, Pro4]NPY greater than  greater than NPY13-36. 2) The Y5 receptor was negatively coupled to cAMP accumulation, as had been proposed for the xe2x80x9catypical Y1xe2x80x9d receptor. 3) Peptides activated the Y5 receptor with a rank order of potency identical to that described for the feeding response. 4) The reported feeding xe2x80x9cmodulatorxe2x80x9d [D-Trp32]NPY bound selectively to the Y5 receptor and subsequently activated the receptor. 5) Both the Y5 and the xe2x80x9catypical Y1xe2x80x9d receptors were sensitive to deletions or modifications in the midregion of NPY and related peptide ligands. These data support the identity of the Y5 receptor as the previously described xe2x80x9catypical Y1xe2x80x9d, and furthermore indicate a role for the Y5 receptor as a potential target in the treatment of obesity, metabolism, and appetite disorders.
The treatment of disorders or diseases associated with the inhibition of the Y5 receptor subtype, especially diseases caused by eating disorders like obesity, bulimia nervosa, diabetes, dislipidimia, may be effected by administration of compounds which bind selectively to the Y5 receptor and inhibit the activation of the Y5 receptor. Furthermore, any disease states in which the Y5 receptor subtype is involved, for example, memory loss, epileptic seizures, migraine, sleep disturbance, pain, and affective disorders such as depression and anxiety may also be treated using compounds which bind selectively to the Y5 receptor.
This invention provides a method of modifying a subject""s feeding behavior which comprises administering to the subject a compound which is a Y5 receptor agonist or antagonist in an amount effective to alter the subject""s consumption of food and thereby modify the subject""s feeding behavior.
This invention also provides a method of treating a subject""s feeding disorder which comprises administering to the subject a non- peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject""s Y5 receptor, wherein the binding of the compound to the human receptor is characterized by a Ki less than 100 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
Additionally, this invention provides a method of treating a subject""s feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject""s Y5 receptor, wherein the compound""s binding to the human Y5 receptor is characterized by a Ki less than 10 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
This invention further provides a method of treating a subject""s feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject""s Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a Ki less than 100 nanomolar when measured in the presence of 125I-PYY in a predetermined amount; a nd (b) the binding of the compound to any other human Y-type receptor is characterized by a Ki greater than 1000 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
This invention also provides a method of treating a subject""s feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject""s Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a Ki less than 1 nanomolar when measured in the presence of 125I-PYY in a predetermined amount; and (b) the compound""s binding to any other human Y-type receptor is characterized by a Ki greater than 100 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
This invention further provides a method of treating a subject""s feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist effective to increase the activity of the subject""s Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a Ki less than 1 nanomolar when measured in the presence of 125I-PYY in a predetermined amount; and (b) the binding of the compound to any other human Y-type receptor is characterized by a Ki greater than 25 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
This invention provides a method of treating a subject""s feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject""s Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a Ki less than 0.1 nanomolar when measured in the presence of 125I-PYY in a predetermined amount; and (b) the binding of the compound to any other human Y-type receptor is characterized by a Ki greater than 1 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
This invention further provides a method of treating a subject""s feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject""s Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a Ki less than 0.01 nanomolar when measured in the presence of 125I-PYY in a predetermined amount; and (b) the binding of the compound to any other human Y-type receptor is characterized by a Ki greater than 1 nanomolar when measured in the presence of 125I-PYY in a predetermined amount.
Additionally, this invention provides an isolated nucleic acid encoding a Y5 receptor. This invention also provides an isolated Y5 receptor protein. This invention provides a vector comprising the above-described nucleic acid.
This invention also provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to the DNA encoding the human Y5 receptor as to permit expression thereof designated pcEXV-hY5 (ATCC Accession No. 75943). This invention further provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to the DNA encoding the rat Y5 receptor as to permit expression thereof designated pcEXV-rY5 (ATCC Accession No. 75944).
This invention provides a mammalian cell comprising the above-described plasmid or vector.
This invention also provides a nucleic acid probe comprising a nucleic acid of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid encoding a Y5 receptor.
Additionally, this invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding a Y5 receptor so as to prevent translation of the mRNA.
This invention also provides an antibody directed to a Y5 receptor.
This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce activity of a human Y5 receptor by passing through a cell membrane and binding specifically with mRNA encoding a human Y5receptor in the cell so as to prevent its translation and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
This invention also provides a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of a human Y5 receptor and a pharmaceutically acceptable carrier. This invention further provides a pharmaceutical composition comprising an amount of an agonist effective to increase activity of a Y5 receptor and a pharmaceutically acceptable carrier. This invention further provides the above-described pharmaceutical composition which comprises an amount of an antibody effective to block binding of a ligand to the Y5 receptor and a pharmaceutically acceptable carrier.
This invention additionally provides a transgenic nonhuman mammal expressing DNA encoding a human Y5 receptor.
This invention also provides a method for determining whether a ligand can specifically bind to a Y5 receptor which comprises contacting a plurality of cells transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the ligand under conditions permitting binding of ligands to such receptor, detecting the presence of any such ligand specifically bound to the Y5 receptor, and thereby determining whether the ligand specifically binds to the Y5 receptor.
This invention further provides a method for determining whether a ligand is a Y5 receptor agonist which comprises contacting a cell transfected with and expressing nucleic acid encoding a human Y5 receptor with the ligand under conditions permitting activation of the Y5 receptor, detecting an increase in Y5 receptor activity, and thereby determining whether the ligand is a human Y5 receptor agonist.
This invention provides a method for determining whether a ligand is a Y5 receptor antagonist which comprises contacting a cell transfected with and expressing DNA encoding a Y5 receptor with the ligand in the presence of a known Y5 receptor agonist, such as PYY or NPY, under conditions permitting the activation of the Y5 receptor, detecting a decrease in Y5 receptor activity, and thereby determining whether the ligand is a Y5 receptor antagonist.
This invention further provides a method of screening a plurality of chemical compounds not known to bind to a Y5 receptor to identify a compound which specifically binds to the Y5 receptor, which comprises (a) contacting a cell transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction from a cell extract of such cells, with a compound known to bind specifically to the Y5 receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the Y5 receptor, under conditions permitting binding of compounds known to bind the Y5 receptor; (c) determining whether the binding of the compound known to bind to the Y5 receptor is reduced in the presence of the compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the Y5 receptor of each compound included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the Y5 receptor.
This invention also provides a method of screening a plurality of chemical compounds not known to activate a Y5 receptor to identify a compound which activates the Y5 receptor which comprises (a) contacting a cell transfected with and expressing the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the plurality of compounds not known to bind specifically to the Y5 receptor, under conditions permitting activation of the Y5 receptor; (b) determining whether the activity of the Y5 receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the Y5 receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the Y5 receptor.
This invention further provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a Y5 receptor to identify a compound which inhibits the activation of the Y5 receptor, which comprises (a) contacting a cell transfected with and expressing the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the plurality of compounds in the presence of a known Y5 receptor agonist, under conditions permitting activation of the Y5 receptor; (b) determining whether the activation of the Y5 receptor is reduced in the presence of the plurality of compounds, relative to the activation of the Y5 receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the Y5 receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the Y5 receptor.
Additionally, this invention provides a process for identifying a chemical compound which specifically binds to a Y5 receptor, which comprises contacting nonneuronal cells expressing on their cell surface the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the Y5 receptor.
This invention also provides a process involving competitive binding for identifying a chemical compound which specifically binds to a Y5 receptor which comprises separately contacting nonneuronal cells expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the Y5 receptor, a decrease in the binding of the second chemical compound to the Y5 receptor in the presence of the chemical compound indicating that the chemical compound binds to the Y5 receptor.
This invention further provides a process for determining whether a chemical compound specifically binds to and activates a Y5 receptor, which comprises contacting nonneuronal cells producing a second messenger response and expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitable for activation of the Y5 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in second messenger response in the presence of the chemical compound indicating that the chemical compound activates the Y5 receptor.
This invention also provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a Y5 receptor, which comprises separately contacting nonneuronal cells producing a second messenger response and expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with both the chemical compound and a second chemical compound known to activate the Y5 receptor, and with only the second chemical compound, under conditions suitable for activation of the Y5 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in second messenger response in the presence of both the chemical compound and the second chemical compound indicating that the chemical compound inhibits activation of the Y5 receptor.
This invention additionally provides a method of treating a subject""s abnormality, wherein the abnormality is alleviated by the inhibition of a Y5 receptor which comprises administering to a subject an effective amount of Y5 receptor antagonist. This invention also provides a method of treating a subject""s abnormality wherein the abnormality is alleviated by the activation of a Y5 receptor which comprises administering to a subject an effective amount of a Y5 receptor agonist.
This invention further provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific allelic form of a human Y5 receptor which comprises: a. obtaining DNA from a subject to be tested; digesting the DNA with restriction enzymes; c. separating the resulting DNA fragments; d. contacting the fragments with a detectably labeled nucleic acid probe capable of specifically hybridizing with a sequence uniquely present within the sequence of a nucleic acid molecule encoding the allelic form of the human Y5 receptor; and e. detecting the presence of labeled probe from the subject to be tested, the presence of such hybridized probe indicating that the subject is predisposed to the disorder.
This invention also provides a method of preparing the isolated Y5 receptor which comprises: a. inserting nucleic acid encoding Y5 receptor in a suitable vector which comprises the regulatory elements necessary for expression of the nucleic acid operatively linked to the nucleic acid encoding a Y5 receptor; b. inserting the resulting vector in a suitable host cell so as to obtain a cell which produces the Y5 receptor; c. recovering the receptor produced by the resulting cell; and d. purifying the receptor so recovered.